Abstract

Ethanol in millimolar concentrations inibited the p-hydroxylation of aniline, a type II compound, but had no effect on the N-demethylation of ethylmorphine, a type I compound. The inhibitory effect of the ethanol on aniline p-hydroxylation was shown to occur at some stage beyond the reduction of cytochrome P-450. Aniline p-hydroxylation was inhibited competitively by all the primary alcohols from ethanol through heptanol, and the inhibitory potency of the alcohols increased with increasing carbon chain length. The increment in the free energy of binding,-0.48 kcal/mole/CH2 group, provided evidence for an alcohol hydrophobic binding site in either the microsomal membrane or the protein. Studies with the fluorescent probe 1-anilino-8-naphthalenesulfonate suggested that the hydrophobic catalytic site resides in the protein. The first requirement for inhibition of aniline p-hydroxylation appeared to be hydrogen bond formation between the hydroxyl group of the alcohol and the enzyme. Steric requirements were also shown to be important. A good correlation between inhibitory potency and logarithm of the partition coefficient was observed for the straight-chain alcohols, but this no longer held for branched-chain compounds. An analysis of the inhibitory data by the method of Hansch showed this system to be one of the most sensitive ascribed to the effects of alcohols.

ACKNOWLEDGMENTS We should like to thank Virginia R. Kickertz for technical assistance, and Dr. Patrick E. Hanna for his helpful views on structure-activity relationships.